Urinary tract infection (UTI) is the second most common infectious disease and causes significant patient morbidity and economic impact. Approximately half of all women will suffer a UTI during their lifetime, and about 25% of those will suffer recurrent UTIs (rUTI), suggesting differences in host susceptibility between patients. Uropathogenic E. coli (UPEC) cause approximately 90% of community-acquired UTIs, but diverse bacteria such as Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae are often associated with complicated UTIs. Since UPEC and other bacteria are now known to establish stable intracellular reservoirs within bladder uroepithelial cells in mice and men, an emerging paradigm is that such intracellular bacterial reservoirs underlie rUTI. Little is known of adaptive immunity in the bladder, and previous attempts at vaccines for UTI have proven marginally successful. We recently developed a murine re-infection model of UTI to characterize immune responses to UPEC, based on infection with the prototypical UPEC isolate NU14 that is introduced into the bladder lumen of mice via catheter. Using this model, we demonstrated that both T cells and antibodies mediate protective immunity against NU14 infection. In parallel studies, we also identified an NU14 mutant in the waaL gene that is highly attenuated for infection of the urinary tract. Preliminary studies demonstrate that this NU14 mutant confers significant protection of both the bladder and kidneys against subsequent challenge with either wild type NU14 or phylogenetically diverse UPEC strains and suggest an enhanced capacity to eradicate intracellular UPEC reservoirs. Therefore, we hypothesize that waaL mutations of pathogenic bacteria represent novel vaccines for protecting the bladder from rUTI by eliciting enhanced cell-mediated responses capable of eliminating intracellular reservoirs. To test this hypothesis, we will pursue three aims. In Aim 1, we will determine components of protective immunity induced by NU14-?waaL. In Aim 2, we will confirm the generalizability of waaL vaccines to the complicated UTI pathogens Proteus, Klebsiella, and Pseudomonas. In Aim 3, we will quantify immune responses induced by administration of waaL vaccines at extra-urinary sites. Thus, our study is both highly responsive to the RFA and paves the way for novel vaccines to protect against UTI. Millions of patients in the U.S. suffer urinary tract infection, causing significant morbidity and burden on the healthcare system. Despite the high prevalence of infection, little is known about the bladder immune response, and no effective vaccines exist to prevent infection. This project will characterize immune responses to a novel class of vaccines to treat urinary tract infection.